Computational protocol: Excessive HDAC activation is critical for neurodegeneration in the rd1 mouse

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Protocol publication

[…] HDAC activity assays were performed on cryosections of 4% PFA P11 fixed eyes. The assay is based on an adaptation of the Fluor de Lys Fluorescent Assay System (Biomol, Hamburg, Germany). Retinal sections were exposed to different concentrations of Fluor de Lys-SIRT2 deacetylase substrate (Biomol) with 2 or 100 μM TSA (Sigma, Steinheim, Germany), 2 mM NAM (Sigma), and 500 μM NAD+ (Biomol) in assay buffer (50 mM Tris/HCl, pH 8.0; 137 mM NaCl; 2.7 mM KCl; 1 mM MgCl2; 1 mg/ml BSA) and 0.1% NP40 and 0.5 × Developer II (Biomol). Images were captured at indicated time points with a fixed exposure time of 2 s using a Zeiss (Jena, Germany) Axiophot microscope ( × 20 magnification) and Zeiss Axiovision 4.2 software; densitometry was performed with the aid of ImageJ (National Institutes of Health, Bethesda, MD, USA). The reaction rate was determined in at least four independent experiments, as the slope of the linear regression fitted to the data points in the linear range of a time course, using SigmaPlot (Systat software, Erkrath, Germany) software. For the calculation of Km values, the slope of time courses with different substrate concentrations was plotted against the corresponding substrate concentrations; a hyperbolic regression fitted to the data points revealed Km values and Vmax.In situ deacetylation activity was determined on retinal sections incubated for 3 h with 200 μM Fluor de Lys-SIRT2 deacetylase substrate in assay buffer with/without inhibitors. Sections were then washed in PBS and fixed in methanol at −80 °C for 20 min. × 0.5 developer (Biomol) in assay buffer was applied and pictures taken immediately in an Apotome microscope (Zeiss), at × 20 using the DAPI filter. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Retinal Degeneration, Retinitis Pigmentosa, Neurodegenerative Diseases
Chemicals Poly Adenosine Diphosphate Ribose